J. Mater. Environ. Sci. 7 (12) (2016) 4608-4613
ISSN : 2028-2508
CODEN: JMESCN
Radi et al.
Validation of a Method for Simultaneous Determination of Acetaminophen
and Caffeine by HPLC in Different Pharmaceutical
Forms: Tablet, Capsule and Sachet
*
1
M. Radi1, Y. Ramli2 , M. El Karbane1, S. Marzak3, K. Bougrin3,
K. El Bourkadi4, F. Ouazzani Chahdi4, S. Issmaili1,K. Bakhous1, A. Ben Ali1
National Medicines Control Laboratory, Rue LamfadalCharkaouiMadinat Al Irfane BP 6206/ Rabat - Morocco.
Medicinal Chemistry Laboratory, Faculty of Medicine and Pharmacy, Mohammed V University - Rabat –Morocco
3
Laboratoire de Chimie des Planteset de Synthèse Organique et Bioorganique, Faculté des Sciences, Mohammed V
University - Rabat –Morocco
4
Laboratoire de Chimie Organique Appliquée, Université Sidi Mohamed Ben Abdallah, Faculté des Sciences et Techniques
2
Received 08 Feb 2016, Revised 06 Apr 2016, Accepted 15 Apr 2016
*Corresponding author. E-mail:[email protected] (Y.Ramli)
Abstract
A simple, fast, economical, accurate, precise and reproducible RP – HPLC method was developed for the
simultaneous estimation of acetaminophen (AAPH) and caffeine (CAF) in starting materialand pharmaceutical
dosage forms. The method was validated in terms of specificity, linearity, precision accuracy, and robustness. The
proposed method’s results were found to be satisfactory and are suitable for determination of acetaminophen and
caffeine for routine quality control of drugs in formulations.
Keywords:acetaminophen, caffeine, HPLC, validation.
1. Introduction
Acetaminophen (AAPH), in figure 1, is the active ingredient of many drug specialties of the class of nonsalicylate antipyretic analgesics. Chemically it is a N-(4-hydroxyphenyl)acetamide. It is indicated for the
symptomatic treatment of fever and low to moderate pain, alone or in association with other analgesics. In
contrast to non-steroidal anti-inflammatory drugs and in particular aspirin, it is devoid of anti-inflammatory
properties and does not act on platelet aggregation. Caffeine (CAF), chemically described as 1,3,7-trimethyl-1Hpurine-2,6(3H,7H)-dione (Fig. 2) is an alkaloid of the methylxanthine family, present in many foods, which acts
as a stimulant psychotrope and as a mild diuretic.
CH3
H
N
O
N
N
N
O
CH 3
HO
H 3C
Figure 1: Structure of acetaminophen (AAPH)
O
Figure 2: Structure of caf f eine (CAF)
Acetaminophen and caffeine-based medicines are available in different pharmaceuticalforms; for example:
Claradol 500mg Caffeine (Bayer Health Care), Exidol (Galephar), Theinol (Bailly-Creat). Currently,
measurement of these molecules in the finished products is done through various methodssuch as UV, HPLC …
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J. Mater. Environ. Sci. 7 (12) (2016) 4608-4613
ISSN : 2028-2508
CODEN: JMESCN
Radi et al.
[1-17]. Hence, the present investigation was aimed at developing a fully validated HPLC-PDA(Photodiode Array)
method for the simultaneous estimation of acetaminophen and caffeine in different pharmaceutical forms.
We propose a simple and rapid analytical method for the determination of these active ingredients in these
pharmaceuticalforms[19,20]. The aim of such a move is to help laboratories and Drug specialist’s pharmaceutical
industry, to reduce the time and cost of analysis and subsequently minimize chemical releases.
In this work, we developed and validated according to ICH Q2B guidelines strategy [21] (International
Conference on Harmonization), a simple and rapid RP-HPLC method. It is specific, linear, accurate, precise and
robust.
2. Experimental
2.1. Apparatus:
The chromatographic systems used is constituted by a Waters 2695 pump, an auto sampler and a Waters 2998
PDA detector. Spectra Manager software and Empower Software data registration were used for all
absorbancemeasurements.TheMettler Toledo scale was manufactured Switzerland.
2.2. Reagents and standards:
The standard used for the determination of acetaminophen is a working standard having a purity of 100.5% and
the water content of 0.1% and that of caffeine was 99.8% purity and water content is 0.11%. The only reagent
used is methanol which is HPLC grade was supplied from Sigma - Aldrich (Germany).
The placebo used in the validation process consists of the usual excipients present in the commercial formulation:
povidone, colloidal anhydrous silica, magnesium stearate, sodium saccharinate, gelatine capsule, lactose and talc.
2.3. Chromatographic conditions:
The chromatographic conditions are gathered in the table 1.The mobile phase was filtered through a 0.45-μm
Millipore filter and degassed by vacuum prior to use.
Table1: Chromatographic conditions of the method
Symmetry RP18 Column, 150 mm × 4.6 mm, 3.5µm.
Column
Flow rate
1 ml/min
Temperature
25°C
Wavelength
275nm
Injection volume
10 µl
Mobile phase
(Methanol / Distilled water): (30% / 70%)vol/vol
Dilution medium
Distilled water
Sample stability
24 hours at room temperature
3. Results and discussion
3.1 The specificity of the chromatographic method:
The specificity of the methodwasconfirmed by the absence of potentialinterferencecaused by the excipients
withacetaminophen and caffeine, by comparing the chromatograms of the blank, placebo, active ingredientalone
(AIA), and that of the reconstitutedpharmaceuticalform (FPR) (Figure 2a)
The purity angles of acetaminophen and caffeinepeaks are lowerthan the threshold (AAPH : 1.340 < 2.476; CAF:
0.281 < 0.285) (Figure 2b).This shows that the methodis capable of easilyassayedacetaminophen and caffeine in
the presence of these excipients.
3.2 Linearity
The linearity of the method was determined by preparing 3 series of five minimum concentrations (70%, 85%,
100%, 115%, 130%) of the target concentration (500μg/mL of acetaminophen and 50μg/ml of caffeine) of the
active ingredients alone (AIA) and reconstituted pharmaceutical form (FPR) (Fig. 3).
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J. Mater. Environ. Sci. 7 (12) (2016) 4608-4613
ISSN : 2028-2508
CODEN: JMESCN
Radi et al.
Figure 2a: Chromatograms of Blank, Placebo, AIA and FPR.
Figure 2b: Chromatographicpurities of AAPH and CAF
Figure3: Chromatograms of the linearity study of the method
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J. Mater. Environ. Sci. 7 (12) (2016) 4608-4613
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CODEN: JMESCN
Radi et al.
The average of each injection zone and graphing the averagepeak relative to the actual concentration of each
solution (Figures 4 and 5).The regression equations of AIAand FPR are linear for acetaminophen and caffeine
(Table 2 and Figures 3, 4).
Figure 4: Linearity of CAF
Figure 5: Linearity of AAPH
Table 2: Study of the linearity of the method.
CAF
Regression
equations
Slope : a
Intercept: b
Correlation
coefficient: r2
Confidence
interval of a
Confidence
interval of b
AAPH
PAS
y=57484x + 62286
FPR
y=57401x + 42325
57484
62286
0.994
57401
42325
0.995
PAS
y=16839x + 101585
FPR
y=16771x + 66304
16839
10158
0.995
16771
66304
0.999
Max=60069.47
Min=54938.28
Max=59738.00
Min= 55114.96
Max=17542,79
Min= 16138.53
Max=17039.59
Min=16502.92
Max=127479.85
Min= -3883.28
Max=101105.75
Min= -17720.41
Max= 281074.85
Min= -78787.95
Max=134809.50
Min= -2451.48
3.3 Precision (repeatability and intermediate precision).
The repeatability and the intermediateprecisionwerevalidated as described in the ICH guidelines Q2B [16] by
performing 3 series (3 differentoperators) of six sampleseachcontaining 100% of active ingredients in a
reconstitutedpharmaceuticalform. (Figure 6).
Figure 6:Chromatograms of the repeatability study of the method.
The relative standard deviations of repeatability and intermediateprecision of the two active ingredients
are lessthan 2% (Table 3), and wecanconcludethat the analyticalmethodchosenisprecise.
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J. Mater. Environ. Sci. 7 (12) (2016) 4608-4613
ISSN : 2028-2508
CODEN: JMESCN
Radi et al.
Table3: Study of the precision of the method.
CAF
Repeatability
RSDr = 0.95%
Intermediateprecision
RSDIP = 0.89%
AAPH
RSDr = 0.57%
RSDIP = 0.53%
3.4 Accuracy
The accuracy of the methodwasdetermined on 3 series (3 different operators) of their constituted pharmaceutical
form having five concentration levels (70%, 85%, 100%, 115% and 130%) of the target concentration. The
averagerecovery of the two active ingredients and the confidence interval of the measurement are reported in
Table 4. They are all included in the standard values fixed by the EuropeanPharmacopoeia (95% and 105%),
therefore the method of assay is accurate.
Table4: Study of the accuracy of the method.
CAF
Average recovery
99.18%
Confidence interval
[98.19 - 100.17]
AAPH
98.93%
[98.52 – 99.34]
3.5. Robustness
The robustness of the method was studied by changing several experimental parameters: the changes in the
method such as changing the eluant flow rate of 0.1 mL/min that is to say a value of 0.9 ml/min and 1.1 ml/min),
the temperature of the column (5°C of the set point namely : 20°C and 30°C), the composition of the mobile
phase (methanol/distilled water) (25/75% and 35/65%), the detection wavelength (272 and 278 nm) and different
column trademarks (Table 5). From the results of the number of theoretical plates, asymmetry factor, resolution,
content and RSD, due to the variation of these parameters, we can conclude that the method is robust.
Table 5: Robustness of the method.
Number of
TheoreticalPlates
N > 3000
AAPH
CAF
Temperature20°C
9580
11080
Temperature25°C
8980
10360
Temperature30°C
11900
11920
Mobile phase 75/25% 10670
12070
Mobile phase 70/30% 11900
10360
Mobile phase 65/35% 8930
9910
10890
11030
Flow = 0.9 ml/min
11900
10360
Flow = 1.0 ml/min
10620
11290
Flow = 1.1ml/min
λ= 278nm
8990
10220
λ= 275nm
11900
10360
λ= 272nm
8980
10240
Waters Column
11900
10360
SunFireColumn
10850
15785
KromasilColumn
9990
11600
Asymmetry
Factor
1.1%< F< 1.5%
AAPH
CAF
1.19
1.18
1.17
1.17
1.18
1.15
1.15
1.14
117
1.17
1.20
1.22
1.16
1.15
1.17
1.17
1.15
1.15
1.10
1.17
1.17
1.17
1.10
1.18
1.17
1.17
1.05
1.15
1.32
1.36
Resolution
> 3.5
Content
(95% to 105%)
RSD
< 2.5%
4.9
5.0
4.9
7.5
5.0
4.1
5.0
5.0
5.0
5.0
5.0
5.0
5.0
16.8
14.1
AAPH
CAF
99.51
99.35
99.85 101.33
99.92
98.73
99.17
97.73
99.85
101.33
99.13
97.43
98.82
99.54
99.85
101.33
100.00
99.35
101.45 102.02
99.85
101.33
103.53 102.07
99.85
101.33
103.39
99.62
99.54
98.49
AAPH CAF
0.2
1.4
0.4
2.2
0.6
1.1
1.8
0.4
2.1
1.4
Though our study of validation of a new method is to determination of acetaminophen and caffeine by HPLC in
different pharmaceutical forms.It is capable of simultaneously dosing acetaminophen and caffeine with a good
resolution (> 3.5).
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ISSN : 2028-2508
CODEN: JMESCN
Radi et al.
Besides the mobile phase prepared by 70% distilled water, the retention time is relatively short (less than 4.5min),
which minimizes the amount of chemical discharges. It is an environment-friendly methodand it will save
considerable analysis time.
Conclusion
The proposed RP-HPLC - PDA methodwasvalidatedfully as per International Conference on Harmonization
(ICH) Guidelines, and found to be applicable for routine quality control analysis for the estimation of AAPH and
CAFincombinationusingisocratic mode of elution.
The assaymethodproposed RP-HPLC wasdemonstrated as a simple, rapid and economical.
The mobile phase consisted of 70% of distilled water, simple to prepare and the analysis time islessthan 6 min
consumes lessthan 6 ml of mobile phase, the flow rate is 1 ml/min, the preparation of the samplesiscarried out in
water only.
The validation of the methodisbased on a statisticalstudy (Cochran's test, Student's test, Fisher test, Dixon'stest...).
The methodisspecific, linear, accurate, faithful and robust. Therefore, thismethodcanbeemployed in quality control to
estimate the amount of AAPH and CAF in bulk and in combined dosage forms.This method will be appropriate
for the simultaneous determination of acetaminophen and caffeine in pharmaceutical forms: tablets, capsule and
sachet.
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